Dexrazoxane Protects Cardiomyocyte from Doxorubicin-Induced Apoptosis by Modulating miR-17-5p

The usage of doxorubicin is hampered by its life-threatening cardiotoxicity in clinical practice. Dexrazoxane is the only cardioprotective medicine approved by the FDA for preventing doxorubicin-induced cardiac toxicity. Nevertheless, the mechanism of dexrazoxane is incompletely understood. The aim of our study is to investigate the possible molecular mechanism of dexrazoxane against doxorubicin-induced cardiotoxicity. We established a doxorubicin-induced mouse and cardiomyocyte injury model. Male C57BL/6J mice were randomly distributed into a control group (Con), a doxorubicin treatment group (DOX), a doxorubicin plus dexrazoxane treatment group (DOX+DEX), and a dexrazoxane treatment group (DEX). Echocardiography and histology analyses were performed to evaluate heart function and structure. DNA laddering, qRT-PCR, and Western blot were performed on DOX-treated cardiomyocytes with/without DEX treatment in vitro. Cardiomyocytes were then transfected with miR-17-5p mimics or inhibitors in order to analyze its downstream target. Our results demonstrated that dexrazoxane has a potent effect on preventing cardiac injury induced by doxorubicin in vivo and in vitro by reducing cardiomyocyte apoptosis. MicroRNA plays an important role in cardiovascular diseases. Our data revealed that dexrazoxane could upregulate the expression of miR-17-5p, which plays a cytoprotective role in response to hypoxia by regulating cell apoptosis. Furthermore, the miRNA and protein analysis revealed that miR-17-5p significantly attenuated phosphatase and tensin homolog (PTEN) expression in cardiomyocytes exposed to doxorubicin. Taken together, dexrazoxane might exert a cardioprotective effect against doxorubicin-induced cardiomyocyte apoptosis by regulating the expression of miR-17-5p/PTEN cascade.


Introduction
The incidence of cancer has increased in recent years, and it is speculated that 13.1 million people will die of cancer in 2030 [1]. Doxorubicin (DOX), an anthracycline antibiotic, is deemed to be one of the most effective frontline chemotherapeutic drugs for treating cancers [2]. While doxorubicin has a broad-spectrum anticancer activity, the severe adverse effects, especially life-threatening cardiotoxicity, limit its clinical application [3]. Free radical-mediated myocytes damage is the first and most thoroughly studied mechanism used to explain doxorubicin-induced cardiotoxicity [4]. Excess ROS could result in DNA damage and cardiomyocyte apoptosis [5]. Nevertheless, the precise molecular mechanism of the doxorubicin-induced cardiomyocyte apoptosis still remains poorly defined.
MicroRNAs (miRNAs) are a class of noncoding RNAs about 22 nucleotides in length, which are reported to posttranscriptionally regulate target gene expression by directly binding to 3 ′ -untranslated regions (3 ′ -UTR) of target messenger RNAs [6]. It has been well recognized that a large number of miRNAs participate in regulating doxorubicininduced cardiotoxicity; thus, they could be used as potential cardiotoxicity biomarkers [7]. MiR-17-5p belongs to miR-17 family, which has been confirmed to be involved in the normal development of organisms and the survival and growth of malignant tumor [8]. A study reported that overexpression of miR-17-5p could suppress the inflammation in LPSinduced macrophages [9]. Furthermore, it has been found that miR-17-5p plays the role of oncogene in most tumors, promotes cell proliferation, and inhibits cell apoptosis [10,11]. Moreover, the recent study has shown that miR-17-5p is downregulated in breast cancer patients with epirubicin-(an isomer) induced cardiotoxicity [12]. Based on these findings, we postulate that miR-17-5p may take part in the regulation of doxorubicin-induced cardiotoxicity.
Dexrazoxane (DEX) is the only cardioprotective medicine approved by FDA for preventing anthracyclineinduced cardiac toxicity [13]. Numerous studies have proved that dexrazoxane could chelate iron to decrease the generation of ROS, thus preventing ROS-induced cardiomyocyte apoptosis [14,15]. However, no research that has focused on miRNAs concerning the cardioprotective effect of dexrazoxane.
In this study, we aim to investigate the molecular mechanism of the protective role of dexrazoxane in doxorubicininduced cardiotoxicity and to determine whether miRNAs are involved in this protective effect.  23, revised 1996). The mice (n = 32) were randomly distributed into a control group (Con), a doxorubicin treatment group (DOX), a doxorubicin plus dexrazoxane treatment group (DOX+DEX), and a dexrazoxane treatment group (DEX). DOX+DEX mice were pretreated with 0.1 ml dexrazoxane solutions (200 mg/kg/day, dissolved in 0.167 mol/l sodium lactate solution) 1 h before 10 mg/kg doxorubicin treatment three times a week. DOX mice were injected with the same volume sodium lactate solution and doxorubicin. DEX mice were injected with the same volume dexrazoxane and saline. Con mice were injected with the same volume sodium lactate solution and saline. All of the mice in the four groups were euthanized 7 days after the initial injection of doxorubicin, and the dose of doxorubicin was modified according to previous studies [16][17][18][19][20].

Materials and Methods
2.3. Cardiac Function Assessment. Echocardiography was measured using Vevo 770 and Vevo 2100 (VisualSonics) instruments from Peking University Third Hospital. Fraction shortening (FS) and ejection fraction (EF) were assessed with Vevo Analysis software (version 2.2.3) as previously described [21]. After echocardiography examination, mice were euthanatized by cervical dislocation, and the hearts were collected for cardiac histological analysis.

Cardiac Histological Analysis.
Histology assays were performed with hearts and sections as previously described [22]. The mouse heart tissues were collected and fixed with 4% paraformaldehyde. Tissues were processed as paraffin section and subsequently analyzed by hematoxylin-eosin staining according to the manufacturer's protocol (Sigma-Aldrich). The sections were imaged by microscopy.
2.7. LDH Assay. The lactate dehydrogenase (LDH) concentration was measured using an LDH assay kit according to the manufacturer's manual (Solarbio, Beijing, China) by a routine microtitre plate reader (wavelength: 572 nm). BioMed Research International the dark for 5 min. The cells were viewed using a fluorescence microscope with a blue/cyan emission filter as described previously [24].
2.9. DNA Ladder Assay. Cells were lysed in lysis buffer (10 mM Tris-Cl pH 8.0, 150 mM NaCl, 0.4% SDS, 10 mM EDTA, and 100 g/ml protease K) and incubated at 37°C overnight with gentle agitation. DNA was extracted with phenol/CHCl3/isoamyl alcohol once and CHCl3/isoamyl alcohol twice. DNA fragmentation was detected by loading 10 μg of total DNA onto 2% agarose gel in Tris acetate/EDTA buffer and visualized by ethidium bromide staining as described previously [24].
2.10. Western Blotting Analysis. The protein was extracted from mouse hearts and cardiomyocytes were lysed with cell lysis buffer supplemented with protease and phosphatase inhibitors. The concentration of protein was measured by Pierce BCA Protein Assay Kit. Equivalent protein (10-20 μg) was electrophoresed on sodium dodecyl sulfatepolyacrylamide gels (12%) and transferred to polyvinylidene fluoride (PVDF) membranes. The members were blocked with 5% nonfat milk for 2 h at room temperature. Then the membranes were probed with specific first antibodies(1 : 1000) at 4°C overnight, including the following antibodies:caspase3, Bax, PTEN, NF-κB, p38MAPK, phosphorylated-NF-κB, phosphorylated-p38MAPK, and GAPDH. Membranes were then incubated with secondary antibody (1 : 5000) for 2 h at room temperature. The signals were visualized with an ECL detection reagent. Densitometry analysis was then performed with Image J software (V1.8.0.112).
2.11. Cell Transfection. miRNA oligos were purchased from GenePharma Biotech (Shanghai). Cardiomyocytes were transferred with miR-17-5p mimics, mimics negative control, miR-17-5p inhibitors, or inhibitors negative control using Lipofectamine RNAiMAX (Invitrogen, USA) following the directions provided by the manufacturer. After 24 h, the cells were treated with or without dexrazoxane and doxorubicin for another 24 h.
2.12. RNA Extraction and Real-Time Quantitative PCR. The total RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA, USA). Reverse transcription was performed using a kit from New England Biolabs. The levels of mature miRNA were performed using the QuantStudio3 Real-Time PCR system (Thermo Fisher Scientific, US) with SYBR Green (TaKaRa, Japan) according to the instructions. The data were presented at least three independent experiments.
2.13. 3′UTR Luciferase Assays. The 3′-untranslated region (3 ′ -UTR) of target genes and their mutant variant were synthesized and digested with SacI and XhoI to generate reporter vectors containing miRNA-binding sites (Shengong Co., China). HEK-293A cells were seeded in 96-well plates and cotransfected with luciferase reporter and miR-17-5p mimics using a transfection reagent (Vigofect, Vigorous Biotechnology, China). The cells were harvested 48 h later, and the luciferase activity was detected using the Dual-Luciferase Reporter Assay System (Promega).
2.14. Statistics. All results were analyzed by GraphPad Prism 6 software (GraphPad Software, CA, USA). The data were presented as the mean ± standard error of mean (SEM). Statistical comparisons between two groups were performed by Student's t-test, and among multiple groups were performed by one-way ANOVA followed by a Bonferroni correction. P < 0:05 was considered statistically significant.

Dexrazoxane Mitigates Doxorubicin-Induced Cardiac
Injury In Vivo. To explore the effect of dexrazoxane on doxorubicin-induced cardiotoxicity in vivo, we used doxorubicin-treated mice to establish a heart failure model. We observed that doxorubicin treatment resulted in significant decrease of body weight and increase of heart/body weight ratio compared with control mice, while dexrazoxane pretreatment could mitigate symptoms (Figures 1(a) and 1(b)). Echocardiography analysis of ejection fraction (EF) % and fraction shortening index (FS) % indicated that doxorubicin could induce heart function loss in vivo. However, dexrazoxane treatment could attenuate heart function loss significantly (Figures 1(c) and 1(d)). Hematoxylin-eosin staining showed that a large amount of inflammatory cells were accumulated in the heart tissue, and the structure of heart tissue was disordered in the doxorubicin (DOX) group compared with the control (Con) group. Nevertheless, dexrazoxane significantly decreased inflammatory cell accumulation and preserved the myocardial structure (Figures 1(e) and 1(f)). Together, these data suggested that dexrazoxane has a potent effect on preventing cardiac injury induced by doxorubicin.

Doxorubicin Decreases Cell Viability and Promotes
Cardiomyocyte Apoptosis In Vitro. We used MTT method to measure the viability of primary cardiomyocytes after doxorubicin treatment. As shown in Figure 2(a), doxorubicin treatment reduced cardiomyocyte viability in a concentrationdependent manner. Western blotting analysis showed that active (cleaved) caspase 3 substantially increased in a dosedependent manner after doxorubicin treatment in cardiomyocyte (Figures 2(b) and 2(c)). These results suggested that doxorubicin could reduce myocyte viability and induce cardiomyocyte apoptosis. The moderate injury for cardiomyocyte is 1 μM doxorubicin, when cell viability declined about 30% and cleaved-caspase 3 significantly increased. Therefore, we used 1 μM doxorubicin in the subsequent experiments to generate the in vitro doxorubicin-induced cardiomyocyte toxicity model.

Dexrazoxane Ameliorates Doxorubicin-Induced
Cardiomyocyte Apoptosis. Given that apoptosis plays an important role in doxorubicin-induced cardiotoxicity [25], we subsequently determined the effect of dexrazoxane on cardiomyocyte apoptosis. We treated cardiomyocytes with different concentrations of dexrazoxane prior to doxorubicin exposure. Western blotting showed that the expression of cleaved-caspase 3 in doxorubicin plus dexrazoxane-(DOX +DEX-) treated group was significantly lower than that of 3 BioMed Research International doxorubicin (DOX) alone. When the concentration is 200 μM, the effect of dexrazoxane reached its peak (Figures 3(a) and 3(b)). Thus, we applied 200 μM dexrazoxane to cardiomyocytes before doxorubicin treatment. The MTT assay showed that pretreatment with dexrazoxane could improve the cardiomyocyte viability, which was decreased by doxorubicin (Figure 3(c)). LDH, as another maker of cellular damage, was dramatically enhanced by doxorubicin and reduced obviously in DOX+DEX group (Figure 3(d)). Hoechst staining revealed that DNA condensation and fragmentation was induced by doxorubicin, which is an indicator of apoptosis. Nevertheless, the apoptotic cardiomyocytes were obviously reduced by dexrazoxane (Figures 3(e) and 3(f)). In addition, an occurrence of DNA laddering was discovered in doxorubicin-treated cardiac myocytes, which could be diminished by dexrazoxane (Figure 3(g)). Western blotting also revealed that active (cleaved) caspase 3 and Bax were decreased obviously in the DOX+DEX group compared with DOX group. Moreover, we found that doxorubicin treatment could increase the level of phosphorylated-p38MAPK and phosphorylated-p65, which is an important inflammation signaling pathways, while pretreatment with dexrazoxane could reverse this effect (Figures 3(h)-3(l)). Our study showed that dexrazoxane may protect doxorubicin-mediated cytotoxicity and apoptosis via p38MAPK/NF-κB signaling pathway.
3.4. mir-17-5p Directly Targets PTEN by Interacting with Its 3′UTR. The results in Figure 4(a) showed that several miRNA expressions were analyzed in doxorubicin-treated cardiomyocytes. MiR-17-5p expression change was most obvious in all the genes. Upon analyzing the sequence, miR-17-5p has been highly conserved in mouse, rat, and human ( Figure 4(b)). Next, we tried to identify miR-17-5p direct downstream targets using the target prediction programs TargetScan, miRBase, and PicTar. We analyzed 8 candidate genes with miR-17-5p binding sites in their 3 ′ -UTRs. We synthesized the wild type 3 ′ -UTR and mutated binding sites 3 ′ -UTR of each candidate gene into a pmiRGLO vector and did the dual-luciferase reporter assay to verify whether miR-17-5p directly bounds to these genes. Our data indicated that the phosphatase and tensin homolog (PTEN), an apoptosis-related gene, is a potential molecular target of miR-17-5p in cardiomyocyte (Figure 4(c)). To investigate miR-17-5p's direct target gene, we cotransfected miR-17-5p mimics with the luciferase reporters into HEK293A cells.
The relative luciferase activity of the PTEN 3 ′ -UTR reporter was obviously declined compared with the control vector. However, when the miR-17-5p binding site in the 3 ′ -UTR of PTEN was mutated, the relative luciferase activity of the PTEN 3 ′ -UTR reporter was consistent with the control vector (Figure 4(d)). Western blotting showed that PTEN expression was significantly decreased after miR-17-5p mimic transfection and increased with the miR-17-5p inhibitor transfection in cardiomyocytes (Figures 4(e)-4(h)). Thus, the data suggest that miR-17-5p could directly bind to PTEN 3 ′ -UTR and inhibit its expression.

Discussion
In the present study, we demonstrated that dexrazoxane protects heart function and prevents doxorubicin-triggered apoptosis. Additionally, dexrazoxane could inhibit doxorubicininduced cardiomyocyte apoptosis by regulating the expression of miR-17-5p/PTEN cascade. As far as we know, this is the first report to show that dexrazoxane may protect cardiomyocyte from doxorubicin-induced injury by modulating miRNA expression and its downstream signal pathway.
Apoptosis is known as type I cell death, which plays a key important role in the development and progression of cardiovascular disease [26][27][28]. Apoptosis could be initiated via extrinsic and intrinsic pathways [29]. A large number of

BioMed Research
International studies have proved that cardiomyocyte apoptosis is a vital feature of doxorubicin-induced cardiotoxicity [30]. Consistent with the previous studies [31,32], we observed a significant decrease of heart function in doxorubicin-treated mice, accompanied with an increase of heart/body weight ratio. Moreover, our in vitro experiments confirmed that doxorubicin could reduce cell viability and cause cardiomyocyte apoptosis, evidenced by increasing the expression of cleaved-caspase 3 and Bax.
As mentioned above, dexrazoxane is the only cardioprotective drug approved by the FDA to resist doxorubicininduced cardiac damage [13]. However, the exact protective mechanism of dexrazoxane remains elusive. Multiple mechanisms have been proposed to contribute the protective effects of dexrazoxane. Some researches revealed that dexrazoxane could chelat iron to reduce the generation of ROS [4,33]. Bures et al. [34] found that dexrazoxane could prevent doxorubicin from binding to the topoisomerase 2β complex. Our results showed that dexrazoxane improved cardiac function and blocked cardiomyocyte apoptosis, which was in line with the results of Suzuki et al. [35].
The cardiotoxicity induced by doxorubicin has been associated with inflammatory cytokines, many of which are modulated by mitogen-activated protein kinases (MAPKs) [36]. Many studies have demonstrated that doxorubicin could increase the expression of phosphorylated-p38MAPK and phosphorylated-NF-κB, which play an important role in activation of inflammation [37,38]. The research of Thandavarayan et al. showed that Schisandrin B could prevent doxorubicin-induced inflammation through inhibition of p38MAPK signaling [39]. In addition to regulating inflammation, p38MAPK can also control cell cycle and apoptosis. Ni et al. and Shati et al. [40,41] found that doxorubicin could promote the activation of p38MAPK, thus promoting the occurrence of apoptosis . Similarly, we also found that doxorubicin could active the p38 MAPK and NF-κB, while dexrazoxane could repress this process. These results supposed that dexrazoxane could protect doxorubicin-induced apoptosis and inflammation by p38MAPK/NF-κB signaling pathway.
There has been a great number of evidence supporting that miRNAs may take part in doxorubicin-induced cardiotoxicity [42][43][44]. Moreover, previous studies have manifested that miR-17-5p plays a vital role in tumor cell survival, and it has antiapoptotic properties [45,46]. In this study, the level of miR-17-5p was markedly declined in doxorubicintreated cardiomyocytes, which could be recovered by dexrazoxane, and overexpression of miR-17-5p could reduce doxorubicin-induced apoptosis in cardiomyocytes.
PTEN (phosphatase and tensin homolog on chromosome 10) is a tumor suppressor, which dephosphorylates PIP3, thereby inhibiting the AKT/mTOR pathway [47]. Hu et al. [48] found that decreasing miR-21 could exert antiapoptotic effect by targeting PTEN in rats. In addition, Yuan et al. [49] determined that miR-19b and miR-20a may suppress myeloma cells apoptosis by targeting PTEN. Our studies also confirmed that PTEN is a pro-apoptosis gene. Fang et al. [50]reported that miR-17-5p could induce drug resistance an invasion of ovarian carcinoma cells by targeting PTEN signaling. Lu et al. [51] discovered that long noncoding RNA HOTAIRMI inhibits cell progression by regulating miR-17-5p/PTEN axis in gastric cancer. Consistent with the previous studies, in our work, we verified that PTEN is the target gene of miR-17-5p, and dexrazoxane might alleviate doxorubicin-triggered apoptosis via miR-17-5p/PTEN signal pathway.

Conclusions
In this study, we proved that dexrazoxane prevents doxorubicin-induced cardiotoxicity by ameliorating apoptosis. We demonstrate for the first time that miR-17-5p plays a key role in the cardioprotective effect of dexrazoxane, which may help to better understand the cardioprotection of dexrazoxane, and miR-17-5p could be a potential molecular target in doxorubicin-induced cardiotoxicity treatment in the future. Moreover, the present findings may offer a new insight to implicate novel drug targets and offer new therapeutic strategies to protect against doxorubicin-induced cardiotoxicity.

Data Availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethical Approval
This study was approved by the Animal Care and Treatment Committee of Beijing Hospital Animal Use and Care Committee and Beijing Normal University Animal Use and Care Committee and the Guide for Care and Use of Laboratory Animals (NIH Publication # 85-23, revised 1996).

Disclosure
Xiaoxue Yu and Yang Ruan are co-first authors.